Expression, purification, and characterization of the Kunitz-type proteinase inhibitor domain of the amyloid β-protein precursor-like protein-2

Abstract

In this report we describe the use of the methylotrophic industrial yeast Pichia pastoris as a host system for the large scale production of the Kunitz-type proteinase inhibitor (KPI) domain of the amyloid β-protein precursor-like protein-2 (APLP-2). The expression plasmid for the KPI domain of APLP-2 encoded amino acids 305–364 of the APLP-2 cDNA (Slunt et al. (1994) J. Biol. Chem. 269, 2637–2644). The secreted 60 amino-acid product was purified to homogeneity and biochemically characterized. Amino-acid sequencing of the expressed KPI domain of APLP-2 verified its integrity. The proteinase inhibitory properties of the KPI domain of APLP-2 were compared to those of the KPI domain of proteinase nexin-2/amyloid β-protein precursor (PN-2/AβPP). Both KPI domains potently inhibited trypsin and, to a lesser extent, chymotrypsin, plasmin, and coagulation factors XIa and IXa. However, the KPI domain of APLP-2 was a ≈20-fold less effective inhibitor of coagulation factor XIa compared to the KPI domain of PN-2/AβPP. Similarly, the KPI domain of APLP-2 was a less effective anticoagulant in coagulation based assays than the KPI domain of PN-2/AβPP. These studies indicate that the KPI domains of PN-2/AβPP and APLP-2 form a family of proteinase inhibitors although the former is a better inhibitor of factor XIa and a more potent anticoagulant than the latter.